Article

Engineering carotenoid production in mammalian cells for nutritionally enhanced cell-cultured foods

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Abstract

Metabolic engineering of mammalian cells has to-date focused primarily on biopharmaceutical protein production or the manipulation of native metabolic processes towards therapeutic aims. However, significant potential exists for expanding these techniques to diverse applications by looking across the taxonomic tree to bioactive metabolites not synthesized in animals. Namely, cross-taxa metabolic engineering of mammalian cells could offer value in applications ranging food and nutrition to regenerative medicine or gene therapy. Towards the former, recent advances in meat production through cell culture suggest the potential to produce meat with fine cellular control, where tuning composition through cross-taxa metabolic engineering could enhance nutrition and food-functionality. Here we demonstrate this possibility by engineering primary bovine and immortalized murine muscle cells with prokaryotic enzymes to endogenously produce the antioxidant carotenoids phytoene, lycopene and β-carotene. These phytonutrients offer general nutritive value and protective effects against diseases associated with red and processed meat consumption, and so offer a promising proof-of-concept for nutritional engineering in cultured meat. We demonstrate the phenotypic integrity of engineered cells, the ability to tune carotenoid yields, and antioxidant functionality of these compounds in vitro towards both nutrition and food-quality objectives. Our results demonstrate the potential for tailoring the nutritional profile of cultured meats. They further lay a foundation for heterologous metabolic engineering of mammalian cells for applications outside of the clinical realm.

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... The sensory experience of meat generally involves taste and aroma from the Maillard reaction and lipid oxidation reactions, color from heme proteins, and a tender, juicy texture [85]. Techniques have begun to be studied in modifying the nutritional profile of cultured meat cell lines, and could also be applied to alter the taste and aroma of cultured meat by variation in the production of different flavor compounds within cells [88]. Cultured meat's color is generally paler than conventional meat because the expression of the heme protein myoglobin is suppressed at ambient oxygen conditions. ...
... Other cultured meats could be engineered for desired differences from the nutritional profile of conventional meat. As an initial exploration of nutritional engineering for cultured meat, Stout et al. have incorporated a biosynthetic pathway for carotenoids, a class of antioxidants native to some plants but not to animals, into primary bovine satellite cells (BSCs) [88]. They demonstrated that these carotenoid-producing BSCs showed antioxidant capacity and reduced lipid oxidation, potentially increasing the nutritional value of meat and reducing the link between red meat consumption and colorectal cancer [88]. ...
... As an initial exploration of nutritional engineering for cultured meat, Stout et al. have incorporated a biosynthetic pathway for carotenoids, a class of antioxidants native to some plants but not to animals, into primary bovine satellite cells (BSCs) [88]. They demonstrated that these carotenoid-producing BSCs showed antioxidant capacity and reduced lipid oxidation, potentially increasing the nutritional value of meat and reducing the link between red meat consumption and colorectal cancer [88]. There is a wealth of possible research in tuning the nutritional characteristics of cultured meat, even including edible therapeutics [88]. ...
Article
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The need to produce immortal, food-relevant cell lines is one of the most pressing challenges of cellular agriculture, the field which seeks to produce meat and other animal products via tissue engineering and synthetic biology. Immortal cell lines have a long and complicated story, from the first recognized immortal human cell lines taken from Henrietta Lacks, to today, where they are used to assay toxicity and produce therapeutics, to the future, where they could be used to create meat without harming an animal. Although work in immortal cell lines began more than 50 years ago, there are few existing cell lines made of species and cell types appropriate for cultured meat. Cells in cultured meat will be eaten by consumers; therefore, cultured meat cell lines will also require unique attributes not selected for in other cell line applications. Specifically, cultured meat cell lines will need to be approved as safe for consumption as food, proliferate and differentiate efficiently at industrial scales, and have desirable taste, texture, and nutrition characteristics for consumers. This paper defines what cell lines are needed, the existing methods to produce new cell lines and their limitations, and the unique considerations of cell lines used in cultured meat.
... Similarly, a production cell population might be chosen or genetically engineered for their rapid division and resistance to senescence (e.g., Wang et al., 2019), resilience under certain environmental conditions, or other desirable features. The use of genetic modification can optimize cell nutrient efficiency, growth, and adaptation to synthetic growth media, or improve final product characteristics, such as nutrition, taste, and texture (Ben-Arye et al., 2020;Lee et al., 2016;Rubio et al., 2019;Stout et al., 2020). ...
... These alterations may be employed to reduce adverse human responses; for example, for those with the meat allergy alpha-gal syndrome, removal of alpha-gal sugars on the surface of cells reduces the chance of allergic reaction to meat products (U.S. Food and Drug Administration, 2020). The novel expression or upregulation of proteins or bioactive metabolites not normally synthesized in animals may improve nutritional value and food quality, such as antioxidant carotenoids (Stout et al., 2020). However, an assessment of whether these expression products are safe for human consumption is needed. ...
... A molecular and biochemical analysis of cell-cultured products can help determine the extent of any differences in the genome and confirm intentional effects or identify unintentional expression of products not normally seen in meat or seafood (Sewalt et al., 2016;Stout et al., 2020). Any expression products may be compared to conventional products to identify any new or increased hazards related to consumption. ...
Article
Full-text available
Cell-cultured meat and seafood offer a sustainable opportunity to meet the world's increasing demand for protein in a climate-changed world. A responsible , data-driven approach to assess and demonstrate safety of cell-cultured meat and seafood can support consumer acceptance and help fully realize the potential of these products. As an initial step toward a thorough demonstration of safety, this review identifies hazards that could be introduced during manufacturing, evaluates applicability of existing safety assessment approaches, and highlights research priorities that could support safe commercialization. Input was gathered from members of the cultured meat and seafood industry, researchers, regulators , and food safety experts. A series of workshops were held with 87 industry representatives and researchers to create a modular manufacturing process diagram , which served as a framework to identify potential chemical and biological hazards along the steps of the manufacturing process that could affect the safety of a final food product. Interviews and feedback on draft documents validated the process diagram and supported hazard identification and evaluation of applicable safety methods. Most hazards are not expected to be novel; therefore, safety assessment methods from a range of fields, such as conventional and novel foods, foods produced from biotechnology, pharmaceuticals, and so forth, are likely to be applicable. However, additional assessment of novel inputs or products with significant differences from existing foods may be necessary. Further research on the safety of the inputs and associated residues, potential for contamination, and development of standardized safety assessment approaches (particularly animal-free methods) is recommended. K E Y W O R D S cultured meat and seafood, hazard, methods, risk assessment, safety.
... Similarly, a production cell population might be chosen or genetically engineered for their rapid division and resistance to senescence (e.g., Wang et al., 2019), resilience under certain environmental conditions, or other desirable features. The use of genetic modification can optimize cell nutrient efficiency, growth, and adaptation to synthetic growth media, or improve final product characteristics, such as nutrition, taste, and texture (Ben-Arye et al., 2020;Lee et al., 2016;Rubio et al., 2019;Stout et al., 2020). ...
... These alterations may be employed to reduce adverse human responses; for example, for those with the meat allergy alpha-gal syndrome, removal of alpha-gal sugars on the surface of cells reduces the chance of allergic reaction to meat products (U.S. Food and Drug Administration, 2020). The novel expression or upregulation of proteins or bioactive metabolites not normally synthesized in animals may improve nutritional value and food quality, such as antioxidant carotenoids (Stout et al., 2020). However, an assessment of whether these expression products are safe for human consumption is needed. ...
... A molecular and biochemical analysis of cell-cultured products can help determine the extent of any differences in the genome and confirm intentional effects or identify unintentional expression of products not normally seen in meat or seafood (Sewalt et al., 2016;Stout et al., 2020). Any expression products may be compared to conventional products to identify any new or increased hazards related to consumption. ...
Preprint
Full-text available
Cell-cultured meat and seafood offer a sustainable opportunity to meet the world’s increasing demand for protein in a climate-changed world. A responsible, data-driven approach to assess and demonstrate safety of cell-cultured meat and seafood can support consumer acceptance and help fully realize the potential of these products. As an initial step towards a thorough demonstration of safety, this review identifies hazards that could be introduced during manufacturing, evaluates applicability of existing safety assessment approaches, and highlights research priorities that could support safe commercialization. Input was gathered from members of the cultured meat and seafood industry, researchers, regulators, and food safety experts. A series of workshops were held with 87 industry representatives and researchers to create a modular manufacturing process diagram, which served as a framework to identify potential chemical and biological hazards along the steps of the manufacturing process that could affect the safety of a final food product. Interviews and feedback on draft documents validated the process diagram and supported hazard identification and evaluation of applicable safety methods. Most hazards are not expected to be novel; therefore, safety assessment methods from a range of fields, such as conventional and novel foods, foods produced from biotechnology, pharmaceuticals, etc., are likely to be applicable. However, additional assessment of novel inputs or products with significant differences from existing foods may be necessary. Further research on the safety of the inputs and associated residues, potential for contamination, and development of standardized safety assessment approaches (particularly animal-free methods) is recommended.
... For commercial production, the use of primary cells would require repeat tissue biopsies from animals which could affect process reproducibility due to variations in animal biology or isolated cell populations 6 . Additionally, further engineering or adapting primary cells to optimize cultured meat products (e.g., through enhanced nutrition or tolerance for waste metabolites) is impractical at production scale, due to the need to re-engineer each new biopsied cell population 7,8 . Thus, immortalized cells are required for further cell engineering and optimization, which would have substantial value for cultured meat production processes. ...
... Cloning and transfections DNA for bovine TERT (Accession #NM_001046242.) and CDK4 (Accession # NM_001037594.2) were obtained as open reading frame clones with additional terminal Flag and HA tags, respectively (GenScript, Piscataway, NJ, USA). Genes were inserted into a Sleeping Beauty plasmid which had been previously developed by our group through the modification of the pSBbi-Pur plasmid from Eric Kowarz (Addgene #60523) 7,46 . This new plasmid, termed pSB-Imm-Puro, contained TERT and CDK4 which were separated by the sequence for a self-cleaving T2A peptide 22 , promoted by a CMV promoter, and followed by a bGH PolyA signal. ...
Preprint
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For cultured meat to succeed at scale, muscle cells from food-relevant species must be expanded in vitro in a rapid and reliable manner to produce millions of metric tons of biomass annually. Toward this goal, genetically immortalized cells offer substantial benefits over primary cells, including rapid growth, escape from cellular senescence, and consistent starting cell populations for production. Here, we develop genetically immortalized bovine satellite cells (iBSCs) via constitutive expression of bovine Telomerase reverse transcriptase (TERT) and Cyclin-dependent kinase 4 (CDK4). These cells achieve over 120 doublings at the time of publication and maintain their capacity for myogenic differentiation. They therefore offer a valuable tool to the field, enabling further research and development to advance cultured meat.
... Faced with a proliferation of climate-related disasters, zoonotic disease outbreaks, and human population growth in recent decades, society is now at a crossroads determining how to satisfy the ever-growing demand for quality dietary protein in a way that is responsible and sustainable 1 . Cultivated meat (CM) is gaining notable traction and investment in recent years as a potential solution to this issue 2,3 and as a way to improve on the food safety, organoleptic, and nutritional attributes of conventional meat products [4][5][6] . However, presently, there are numerous technical hurdles to overcome before this food technology can be commercially viable at large scales 7 . ...
... In the present paper, we present a straightforward characterization and comparison of three different cell types that are highly relevant to CM research and development. Murine C2C12s are not necessarily going to be useful in developing larger-scale CM bioprocesses in the longer term as the industry develops, although they are a useful model for bench-scale, early-stage CM research; indeed, they are used in many contemporary publications in the space 4,[19][20][21][22] . Regardless, C2C12s, cMPCs, and cMFBs will not be the only cell types utilized in the CM industry, and the specific cellular behaviors that we observed in our study may not be generalizable to all other CM cell types. ...
Article
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Cell culture media design is perhaps the most significant hurdle currently facing the commercialization of cultivated meat as an alternative source of dietary protein. Since media optimization for a specific culture system requires a significant amount of effort and investment, a major question remaining is whether media formulations can be easily shared across multiple production schemes for cells of different species and lineages. Here, we perform spent medium analysis to compare the specific nutrient utilization of primary embryonic chicken muscle precursor cells and fibroblasts to the murine C2C12 myoblast cell line. We demonstrate that these related cell types have significantly different nutrient utilization patterns collectively and on a per-cell basis, and that many components of conventional media do not appear to be depleted by the cells. Namely, glucose was not consumed as rapidly nor as completely by the chicken muscle precursors compared to other cells overall, and there were significant differences in specific consumption rates for several other key nutrients over the first day of culture. Ultimately, our results indicate that no one medium is likely ideal and cost effective to culture multiple cell types and that novel methods to streamline media optimization efforts will be important for the industry to develop. npj Science of Food (2022) 6:46 ; https://doi.
... For instance, early life-cycle analyses suggest that cultured meat could require >90% less land and >75% less water than conventional beef, while contributing >75% fewer greenhouse gas emissions, >95% less eutrophication, and >90% less particulate matter formation 2,3 . At the same time, cultured meat could improve animal welfare, foodsystem resilience, and human health outcomes [4][5][6] . The challenges that face the successful technological transition of cultured meat to the marketplace stem from the need for production systems that are low-cost, scalable, food-safe, and free of animal-derived inputs 4,7,8 . ...
... Primary bovine satellite cell isolation and maintenance. Primary bovine satellite cells (BSCs) were isolated with methods previously used in our group and based on previously described pre-plating satellite-cell isolation protocols 6,41 . Briefly,~0.5 g of muscle was excised from the semitendinosus of a 14-day-old Simmental calf at the Tufts Cummings School of Veterinary Medicine according to approved methods (IACUC protocol #G2018-36). ...
Article
Full-text available
Cell-cultured meat offers the potential for a more sustainable, ethical, resilient, and healthy food system. However, research and development has been hindered by the lack of serum-free media that enable the robust expansion of relevant cells (e.g., muscle satellite cells) over multiple passages. Recently, a low-cost serum-free media (B8) was described for pluripotent stem cells. Here, B8 is adapted for bovine satellite cells through the addition of a single component, recombinant albumin, which renders it suitable for long-term satellite cell expansion without sacrificing myogenicity. This new media (Beefy-9) maintains cell growth over the entire period tested (seven passages), with an average doubling time of 39 h. Along with demonstrated efficacy for bovine cells, Beefy-9 offers a promising starting-point for developing serum-free media for other meat-relevant species. Ultimately, this work offers a foundation for escaping cultured meat research’s reliance on serum, thereby accelerating the field. A substitution of FBS in culturing of bovine satellite cells for the purpose of culturing meat is presented, addressing both basal media and growth factors in relation to proliferating and differentiating cells.
... Primary bovine satellite cells (BSCs) were seeded on glutenin fibrous scaffolds. The isolation of BSCs was based on previously described methods [40,41]. Briefly, a small muscle excision (~1.0 cm 3 ) was taken from the semitendinosus of a seven-month-old Simmental bull raised at the Tufts Cummings School of Veterinary Medicine using approved protocols (IACUC protocol #G2018-36). ...
... To induce myogenic differentiation, both C2C12 cells and BSCs were cultured for 21 days, and then incubated for 7 days without changing the medium to deplete the serum and induce differentiation as in our prior work [41]. ...
Article
Scaffolds suitable for use in food products are crucial components for the production of cultured meat. Here, wheat glutenin, an inexpensive and abundant plant-based protein, was used to develop 3D porous scaffolds for cultured meat applications. A physical cross-linking method based on water annealing was developed for the fabrication of porous glutenin sponges and fibrous aligned scaffolds. The pore sizes ranged from 50 to 250 μm, with compressive modulus ranges from 0.5 to 1.9 kPa, depending on the percentage of glutenin (2%–5%) used in the process. The sponges were stable in PBS with refrigeration for at least six months after water annealing. The glutenin scaffolds supported the proliferation and differentiation of C2C12 mouse skeletal myoblasts and bovine satellite cells (BSCs) without the need to add specific cell adhesive proteins or other coatings. The low cost and food safe production process avoided the use of toxic cross-linkers and animal-derived extracellular matrix (ECM) coatings, suggesting that this as approach is a promising system for scaffolds useful in cultured meat applications.
... Scaling production technology is still under development, but once produced at scale, cultivated meat promises to provide a protein source that uses fewer environmental resources and poses less public health risk than conventional meat (Landers et al., 2012;Tuomisto et al., 2014;Mattick et al., 2015;Smetana et al., 2015;Tuomisto, 2019;Chriki and Hocquette, 2020). Additionally, cultivated meat technologies are precise, creating the possibility of developing healthier and more nutritious products when compared to conventional meat, such as meat with higher omega fatty acid levels or lower saturated fat and cholesterol (Stout et al., 2020). At this time, cultivated meat is only available for purchase from one restaurant in Singapore. ...
... Although plant-based meat nutritional composition varies widely (Harnack et al., 2021), a 2020 clinical trial found improvements in cardiovascular risk factors in comparison to consumption of conventional meat (Crimarco et al., 2020). The nutritional composition of cultivated meat is still under development and could be either similar or improved in comparison to conventional meat (Stout et al., 2020). Given these differences, nutritional health claims may need to be product-specific. ...
Article
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The purpose of this study was to assess the likelihood of consumer adoption of plant-based and cultivated meat in South Africa as a pathway to a healthy, sustainable, and equitable food supply. We recruited a large sample of South Africans representative across age (18–61), gender, race, and income to participate in an online survey. Participants responded to a range of measures including adoption indicators, estimated yearly intake, motivators for purchasing, desired product characteristics, preferred species, and sociodemographics. We found a high degree of openness to both products. For plant-based meat, 67% were highly likely to try and 59% were highly likely to purchase. For cultivated meat, 60% were highly likely to try and 53% were highly likely to purchase. The highest acceptance was amongst the younger generations: 60% of born-frees, 62% of millennials, and 53% of Gen X were highly likely to purchase plant-based meat and 55% of born-frees, 55% of millennials, and 46% of Gen X were highly likely to purchase cultivated meat. For the general population, we observed that future meat intake was estimated to be split equally among the three meat categories (conventional, cultivated, and plant-based). We found early adopters (those highly likely to purchase) to be quite similar in attitudinal and sociodemographic characteristics in comparison to the general population. The study findings suggest that both plant-based and cultivated meat could be viable market-based options for improving the food system in South Africa, as consumers across all segments of society, and especially amongst the younger population, indicated broad acceptance.
... For instance, limited life-cycle analyses suggest that cultured meat could require >90% less land and >75% less water than conventional beef, while contributing >75% fewer greenhouse gas emissions, >95% less eutrophication, and >90% less particulate matter formation 2,3 . At the same time, cultured meat could improve animal welfare, food security, and human health outcomes [4][5][6] . The challenges that face the successful technological transition of cultured meat to the marketplace stem from the need for production systems that are low-cost, scalable, food-safe, and free of animal-derived inputs 4,7,8 . ...
... Primary bovine satellite cell isolation and maintenance Primary bovine satellite cells (BSCs) were isolated with methods previously used in our group 6 . Briefly, ~0.5 g of muscle was excised from the semitendinosus of a 14-day-old Simmental calf at the Tufts Cummings School of Veterinary Medicine according to approved methods (IACUC protocol #G2018-36). ...
Preprint
Full-text available
Cell-cultured meat offers the potential for a more sustainable, ethical, resilient, and healthy food system. However, research and development has been hindered by the lack of suitable serum-free media that enable the robust expansion of relevant cells (e.g., muscle satellite cells) over multiple passages. Recently, a low-cost serum-free media (B8) was described for induced pluripotent stem cells. Here, we adapt this media for bovine satellite cells and show that the addition of a single component, recombinant albumin, renders B8 suitable for the long-term expansion of cells without sacrificing myogenicity. We show that this new media (Beefy-9) maintains robust cell growth over the entire culture period tested (seven passages) with an average growth rate of 39 hours per population doubling). Along with demonstrated efficacy for bovine cells, this work provides a promising starting-point for developing serum-free media for cultures from other meat-relevant species. Ultimately, this work offers a promising foundation for escaping the reliance on serum in cultured meat research, thereby accelerating the field.
... The primary bovine satellite cells (BSCs) used for this work have been extensively characterized and validated in previous studies 7,23 . To test the ability of OPIs to replace recombinant albumin in serum-free media, BSCs were cultured for four days in either B8 medium without supplementation, B8 medium supplemented with OPIs at a range of concentrations, or Beefy-9 medium (B8 + 0.8 mg/mL recombinant albumin). ...
Preprint
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The development of cost-effective serum-free media is essential for the economic viability of cultured meat. A key challenge facing this goal is high-cost recombinant albumin that is necessary in some available serum-free media formulations. As such, there is substantial interest in finding albumin alternatives which are low-cost, effective, scalable, sustainable, and suitable for food applications. Recently, a serum-free medium termed Beefy-9 was developed for bovine satellite cells (BSCs), which relied on recombinant albumin as a key component to replace fetal bovine serum. Here we alter Beefy-9 by replacing albumin with rapeseed protein isolate, a bulk-protein solution obtained from agricultural waste-streams through simple isoelectric protein precipitation. This new medium, termed Beefy-R, improves BSC growth compared with Beefy-9 while maintaining cell phenotype and myogenicity. These results offer an effective, low-cost, and sustainable alternative to albumin for serum-free culture of muscle stem cells, thereby addressing a key hurdle facing cultured meat production.
... C2C12s and BSCs were seeded on the films with a density of 10,000 cells/cm 2 in 24 ultra-low attachment well plates (Corning™ 3473, USA). To induce myogenic differentiation, both C2C12s and BSCs were cultured for 6 days and then incubated for 6 days without changing the medium to deplete the serum, as described in our previous work [43]. The fixation, permeabilization and blocking of C2C12s and BSCs on films were performed using an Image-iT™ Fixation/Permeabilization Kit (ThermoFisher) according to the manufacturer's instructions. ...
Article
Biomaterial scaffolds are critical components in cultivated meat production for enabling cell adhesion, proliferation, differentiation and orientation. Currently, there is limited information on the fabrication of edible/biodegradable scaffolds for cultivated meat applications. In the present work, several abundant, naturally derived biomaterials (gelatin, soy, glutenin, zein, cellulose, alginate, konjac, chitosan) were formed into films without toxic cross-linking or stabilizing agents. These films were investigated for support of the adhesion, proliferation and differentiation of murine and bovine myoblasts. These biomaterials supported cell viability, and the protein-based films showed better cell adhesion than the polysaccharide-based films. Surface patterns induced cell alignment and guided myoblast differentiation and organization on the glutenin and zein films. The mechanical properties of the protein films were also assessed and suggested that a range of properties can be achieved to meet food-related goals. Overall, based on adherence, proliferation, differentiation, mechanics, and material availability, protein-based films, particularly glutenin and zein, showed the most promise for cultivated meat applications. Ultimately, this work presents a comparison of suitable biomaterials for cultivated meat applications and suggests future efforts to optimize scaffolds for efficacy and cost.
... Cryopreservation of cells has long been necessary in the use of cells in research, for in vitro fertilization, and the increased use of mammalian cells in the production of therapeutics, vaccines, and even food production [1][2][3][4]. Thus, there is a need for cryoprotectants and improved cryopreservation protocols that will enhance the viability of cells stored at low temperatures, and some of these can be found as naturally occurring proteins [5]. ...
Article
Full-text available
Cell cryopreservation is an essential part of the biotechnology, food, and health care industries. There is a need to develop more effective, less toxic cryoprotective agents (CPAs) and methods, especially for mammalian cells. We investigated the impact of an insect antifreeze protein from Anatolica polita (ApAFP752) on mammalian cell cryopreservation using the human embryonic kidney cell line HEK 293T. An enhanced green fluorescent protein (EGFP)-tagged antifreeze protein, EGFP–ApAFP752, was transfected into the cells and the GFP was used to determine the efficiency of transfection. AFP was assessed for its cryoprotective effects intra- and extracellularly and both simultaneously at different concentrations with and without dimethyl sulfoxide (DMSO) at different concentrations. Comparisons were made to DMSO or medium alone. Cells were cryopreserved at −196 °C for ≥4 weeks. Upon thawing, cellular viability was determined using trypan blue, cellular damage was assessed by lactate dehydrogenase (LDH) assay, and cellular metabolism was measured using a metabolic activity assay (MTS). The use of this AFP significantly improved cryopreserved cell survival when used with DMSO intracellularly. Extracellular AFP also significantly improved cell survival when included in the DMSO freezing medium. Intra- and extracellular AFP used together demonstrated the most significantly increased cryoprotection compared to DMSO alone. These findings present a potential method to improve the viability of cryopreserved mammalian cells.
... The direct use of animal muscle and fat cells in cultured meat might also provide a greater resemblance in taste and nutrition to conventional meat when compared to plant based meats [23,29,[43][44][45]. Moreover, control over cell biology during tissue cultivation, as well as the overall production process, allows for the fine tuning of nutritional properties to improve human health, where muscle and fat cells can be engineered to produce vital nutrients such as anti-oxidative carotenoids that would otherwise not be found (or only at low concentrations) in conventional meat [46]. Taken together, these factors suggest that cultured meat production systems may be able to offer healthier, more efficient, and more environmentally compatible options to traditional animal sourced meats. ...
Article
With rising global demand for food proteins and significant environmental impact associated with conventional animal agriculture, it is important to develop sustainable alternatives to supplement existing meat production. Since fat is an important contributor to meat flavor, recapitulating this component in meat alternatives such as plant based and cell cultured meats is important. Here, we discuss the topic of cell cultured or tissue engineered fat, growing adipocytes in vitro that could imbue meat alternatives with the complex flavor and aromas of animal meat. We outline potential paths for the large scale production of in vitro cultured fat, including adipogenic precursors during cell proliferation, methods to adipogenically differentiate cells at scale, as well as strategies for converting differentiated adipocytes into 3D cultured fat tissues. We showcase the maturation of knowledge and technology behind cell sourcing and scaled proliferation, while also highlighting that adipogenic differentiation and 3D adipose tissue formation at scale need further research. We also provide some potential solutions for achieving adipose cell differentiation and tissue formation at scale based on contemporary research and the state of the field.
... The other potential benefits associated with the cultured meat is the controlled modification of the nutritional profile, e.g., by decreasing the saturated fat content or replacing it with omega-3 fatty acids [78]. However, large-scale in vitro cultures require significant effort and cost investment to be maintained, mostly concerning sourcing and preparation of adequate volumes of high-quality nutrient-rich serum, that is now commonly derived from bovine fetuses. ...
Article
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The coronavirus disease 2019 (COVID-19) pandemic is a global health challenge with substantial adverse effects on the world economy. It is beyond any doubt that it is, again, a call-to-action to minimize the risk of future zoonoses caused by emerging human pathogens. The primary response to contain zoonotic diseases is to call for more strict regulations on wildlife trade and hunting. This is because the origins of coronaviruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), as well as other viral pathogens (e.g., Ebola, HIV) are traceable to wild animals. Although COVID-19 is not related to livestock animals, the pandemic increased general attention given to zoonotic viral infections—the risk of which can also be associated with livestock. Therefore, this paper discusses the potential transformation of industrial livestock farming and the production of animal products, particularly meat, to decrease the risks for transmission of novel human pathogens. Plant-based diets have a number of advantages, but it is unrealistic to consider them as the only solution offered to the problem. Therefore, a search for alternative protein sources in insect-based foods and cultured meat, important technologies enabling safer meat production. Although both of these strategies offer a number of potential advantages, they are also subject to the number of challenges that are discussed in this paper. Importantly, insect-based foods and cultured meat can provide additional benefits in the context of ecological footprint, an aspect important in light of predicted climate changes. Furthermore, cultured meat can be regarded as ethically superior and supports better food security. There is a need to further support the implementation and expansion of all three approaches discussed in this paper, plant-based diets, insect-based foods, and cultured meat, to decrease the epidemiological risks and ensure a sustainable future. Furthermore, cultured meat also offers a number of additional benefits in the context of environmental impact, ethical issues, and food security.
... Thirdly, the production process is much more predictable and less time-consuming with several weeks needed for the conversion of nutrients and energy instead of the months or years needed in the case of meat originating from livestock (Bhat et al., 2015). Tissue engineering and cell culturing enable the regulation of nutritional content (e.g., fat) of meat and its enhancement (e.g., by induction or addition of omega-3 fatty acids, carotenoids, etc.) (Stout et al., 2020). Moreover, cultured meat production requires significantly less water and less energy, and its implementation should reduce greenhouse emissions, particularly under the scenario of decarbonized energy generation (Fox, 2009;Lynch and Pierrehumbert, 2019;Tuomisto and Teixeira de Mattos, 2011). ...
Article
The pandemic of the novel coronavirus disease 2019 (COVID-19) has caused a significant burden to healthcare systems, economic crisis, and public fears. It is also a lesson to be learned and a call-to-action to minimize the risk of future viral pandemics and their associated challenges. The present paper outlines selected measures (i.e., monitoring and identification of novel viral agents in animals, limitations to wildlife trade, decreasing hunting activities, changes to mink farming and meat production), the implementation of which would decrease such a risk. The role of viral surveillance systems and research exploring the virus strains associated with different animal hosts is emphasized along with the need for stricter wild trade regulations and changes to hunting activities. Finally, the paper suggests modifications to the meat production system, particularly through the introduction of cultured meat that would not only decrease the risk of exposure to novel human viral pathogens but also strengthen food security and decrease the environmental impacts of food production.
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Cultivated meat may be a more ethical, environmentally friendly, antibiotic-free meat alternative of the future. As of now, one of the main limiting factors for bringing cultivated meat to the market is the high cost of the cell culture medium. Here, we optimize B8/B9 medium - one of the well-established serum free, fully defined medium compositions available for purchase or for preparation in-house. We show several combinations of the growth factors/myokines/hormones, which were able to substantially increase bovine satellite cells’ proliferation rate, as well as treatment schemes which allow to use five to ten times lower concentrations of signaling molecules for the same effect. Additionally, we present two food grade low-price medium stabilizers, one of which exhibits superior stabilization of the B8 medium as compared to recombinant human serum albumin, and allows for its substitution, dropping the price for stabilization to approx. under 0.2% of that used in B9 medium.
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Cultured meat is introduced as a valuable traditional meat equivalent. However, before marketable end products are available, several hurdles need to be overcome. Among others, these issues comprise obtaining an optimal nutritional profile and approaching the texture, the colour and the unique flavour and taste of conventional meat. Furthermore, the impact of processing on these matters is also still subject of future research. Moreover, more profound knowledge on food-safety aspects, like microbial contamination, prions, possible genetically engineered starting material, etc., and ways to reduce such risks will determine the future success of cultured meat products. Undoubtedly, correct terminology and adequate definitions also require further attention, as these form the starting point of legislative/regulatory aspects. This review provides a state-of-the-art overview on nutritional, technofunctional and sensorial properties, and food-safety and legislative/regulatory aspects on cultured meat production. Additionally, the various challenges and future steps of these aspects of cultured meat are highlighted.
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Cultured meat is an emerging technology that is friendly for the environment and animal welfare. As a novel food ingredient, cultured fat is essential for the flavor and nutrition of cultured meat. In this study, we purified adipose progenitor cell (APC) from freshly isolated porcine stromal vessel fraction (SVF) by fluorescence-activated cell sorting (FACS) and identified the transcriptome characteristics of APC by RNA sequencing (RNA-seq). The results showed that APC had characteristics of high-efficiency proliferation and adipogenic differentiation and was distinct from SVF cell in transcriptome profiles. Subsequently, APC was used to prepare cultured fat by 3D bioprinting and to evaluate the differences in fatty acid composition between cultured fat and porcine subcutaneous adipose tissue (pSAT). The results indicated that the fatty acid composition and content of cultured fat had a certain similarity with pSAT; specifically, the content of key monounsaturated fatty acid (MUFA) that create pork flavor in cultured fat, such as C18:1(n-12), C18:1(n-9) and C19:1(n-9)T, were close to that of pSAT. Therefore, this research indicated that APC is a promising candidate cell type for the production of cultured fat.
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Alternative proteins, such as cultivated meat, have recently attracted significant attention as novel and sustainable food. Fat tissue/cell is an important component of meat that makes organoleptic and nutritional contributions. Although adipocyte biology is relatively well investigated, there is limited focus on the specific techniques and strategies to produce cultivated fat from agricultural animals. In the assumed standard workflow, stem/progenitor cell lines are derived from tissues of animals, cultured for expansion, and differentiated into mature adipocytes. Here, we compile information from literature related to cell isolation, growth, differentiation, and analysis from bovine, porcine, chicken, other livestock, and seafood species. A diverse range of tissue sources, cell isolation methods, cell types, growth media, differentiation cocktails, and analytical methods for measuring adipogenic levels were used across species. Based on our analysis, we identify opportunities and challenges in advancing new technology era toward producing “alternative fat” that is suitable for human consumption.
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Cell-cultivated fish offers the potential for a more ethical, sustainable, and safe seafood system. However, fish cell culture is relatively understudied in comparison to mammalian cells. Here, we established and characterized a continuous Atlantic mackerel (Scomber scombrus) skeletal muscle cell line (Mack cells). The cells were isolated from muscle biopsies of fresh-caught fish, with separate isolations performed from two distinct fish. Mack1 cells (cells from the first isolation) were cultured for over a year and subcultured over 130 times. The cells proliferated at initial doubling times of 63.9 hr (+/- 19.1 SD). After a spontaneous immortalization crisis from passages 37-43, the cells proliferated at doubling times of 24.3 hr (+/- 4.91 SD). A muscle phenotype was confirmed through characterization of muscle stemness and differentiation via paired-box protein 7 (PAX7) and myosin heavy chain (MHC) immunostaining, respectively. An adipocyte-like phenotype was also demonstrated for the cells through lipid accumulation, confirmed via Oil Red O (ORO) staining and quantification of neutral lipids. New qPCR primers (HPRT, PAX3B, MYOD1, MYOG, TNNT3A, and PPARG) were tailored to the mackerel genome and used to characterize mackerel cell genotypes. This work provides the first spontaneously immortalized fish muscle cell line for research, ideally serving as a reference for subsequent investigation.
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Cultivating meat from stem cells rather than by raising animals is a promising solution to concerns about the negative externalities of meat production. For cultivated meat to fully mimic conventional meat's organoleptic and nutritional properties, innovations in scaffolding technology are required. Many scaffolding technologies are already developed for use in biomedical tissue engineering. However, cultivated meat production comes with a unique set of constraints related to the scale and cost of production as well as the necessary attributes of the final product, such as texture and food safety. This review discusses the properties of vertebrate skeletal muscle that will need to be replicated in a successful product and the current state of scaffolding innovation within the cultivated meat industry, highlighting promising scaffold materials and techniques that can be applied to cultivated meat development. Recommendations are provided for future research into scaffolds capable of supporting the growth of high‐quality meat while minimizing production costs. Although the development of appropriate scaffolds for cultivated meat is challenging, it is also tractable and provides novel opportunities to customize meat properties. Cultivating meat from cells is a promising solution to the environmental, ethical, health, and food‐security challenges associated with conventional meat production. Cultivated meat will require the development of scalable, low‐cost, and edible or biodegradable scaffolds to support cell growth. This review discusses the unique challenges of cultivated meat scaffolding and highlights promising materials and processing methods worthy of further investigation.
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Cell-cultured meat and seafood offer a sustainable approach to meet the world’s increasing demand for protein in a climate-changed world. A responsible, data-driven approach to assessing and demonstrating safety of cell-cultured meat and seafood can support consumer acceptance and broad distribution; helping to fully realize the potential of these products. A thorough demonstration of food safety requires a description and understanding of the process steps during cell-cultured meat and seafood manufacture to identify potential hazards, their exposure, and related safety requirements related to the inputs, by-products, and ultimately the consumption of the final product. For this review, the authors worked with 50 cultured meat and seafood companies to co-develop a modular manufacturing process diagram, portraying the majority of cell-cultured meat and seafood production processes currently in development. This diagram serves as a framework to identify potential manufacturing hazards and product safety testing requirements. A series of workshops were held with industry representatives and cell-cultured meat researchers to substantiate the accuracy and validity of the diagram. This review identifies potential hazards and where they may occur during product development and manufacture, then evaluates where existing safety assessment approaches, drawn from related fields, may be relevant for cell-cultured meat and seafood products. From this, current knowledge gaps and research priorities were identified regarding the safety of cell-cultured meat and seafood products.
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Laboratory-grown meat has been stuck in the experimental stage. For it to become a commercially viable industry, tissue needs to be grown efficiently at scale. Laboratory-grown meat has been stuck in the experimental stage. For it to become a commercially viable industry, tissue needs to be grown efficiently at scale.
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Skeletal muscle-tissue engineering can be applied to produce cell-based meat for human consumption, but growth parameters need to be optimized for efficient production and similarity to traditional meat. The addition of heme proteins to plant-based meat alternatives was recently shown to increase meat-like flavor and natural color. To evaluate whether heme proteins also have a positive effect on cell-based meat production, bovine muscle satellite cells (BSCs) were grown in the presence of hemoglobin (Hb) or myoglobin (Mb) for up to nine days in a fibrin hydrogel along 3D-printed anchor-point constructs to generate bioartificial muscles (BAMs). The influence of heme proteins on cell proliferation, tissue development, and tissue color was analyzed. We found that the proliferation and metabolic activity of BSCs was significantly increased when Mb was added, while Hb had no, or a slightly negative, effect. Hb and, in particular, Mb application led to a very similar color of BAMs compared to cooked beef, which was not noticeable in groups without added heme proteins. Taken together, these results indicate a potential benefit of adding Mb to cell culture media for increased proliferation and adding Mb or Hb for the coloration of cell-based meat.
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The demand for sustainable foods and an increased consciousness of health and well-being, as well as other societal changes, create opportunities to develop novel foods. However, consumers are programmed from early childhood to prefer familiar foods. We now know that individual variations in disposition determine responses to novelty. Disgust, along with food neophobia and related traits, has been identified as a major barrier to accepting novel food alternatives. In this paper, we present two novel foods trends (meat alternatives and products for health and well-being) as examples of current research. We conclude that successfully launching novel foods require a deep understanding of product perception and the consumer traits that determine rejection or acceptance.
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Tissue engineering is primarily associated with medical disciplines; thus, research has focused on mammalian cells. For applications where clinical relevance is not a constraint, it is imperative to evaluate the potential of alternative cell sources to form tissues in vitro. Specifically, skeletal muscle tissue for bioactuation and cultured foods could benefit from the inclusion of invertebrate cells, due to less stringent growth requirements and versatile features related to biomass. Here, we used a Drosophila muscle cell line to demonstrate the benefits of insect cells relative to those derived from vertebrates. The cells were adapted to serum-free media, transitioned between adherent and suspension cultures, and manipulated via hormones. Furthermore, we analyzed scaffolds to support muscle growth and assayed cellular protein and minerals to evaluate nutrition potential. The insect muscle cells exhibited advantageous growth patterns and hold unique functionality for tissue engineering applications beyond the medical realm.
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Isolating and maintaining the appropriate stem cell for large scale cell culture is essential in tissue engineering or food production. For bovine satellite cells an optimized isolation and purification protocol is lacking and there is also no detailed understanding on the factors that maintain stemness of these cells. Here, we set up a fluorescence-activated cell sorting strategy to enrich bovine satellite cells. We found that p38-MAPK signalling is activated and PAX7 expression is gradually lost during satellite cell proliferation. The p38 inhibitor (SB203580) treatment maintained PAX7 expression but inhibited the fusion of satellite cells in a concentration-dependent way in short-term incubation. The mechanism of p38 inhibition was confirmed by inhibiting canonical p38 signalling, i.e. HSP27. Long-term culture with an appropriate concentration of p38i enhanced the proliferation and PAX7 expression, while the differentiation capacity recovered and was enhanced compared to vehicle control. These studies indicate that bovine satellite cells maintenance depends on cell purity and p38 MAPK signalling. Inhibition of p38 MAPK signaling is a promising strategy to facilitate large scale cell expansion of primary cells for tissue engineering and cultured meat purposes.
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Potato (Solanum tuberosum L.) is the third most widely consumed plant food by humans. Its tubers are rich in starch and vitamin C, but have low or null levels of essential nutrients such as provitamin A and vitamin E. Transformation of potato with a bacterial mini-pathway for β-carotene in a tuber-specific manner results in a “golden” potato (GP) tuber phenotype resulting from accumulation of provitamin A carotenoids (α- and β-carotene) and xanthophylls. Here, we investigated the bioaccessibility of carotenoids and vitamin E as α-tocopherol (αTC) in boiled wild type and golden tubers using in vitro digestion. Golden tubers contained up to 91 μg provitamin A carotenes (PAC)/g D, increased levels of xanthophylls, phytoene and phytofluene, as well as up to 78 μg vitamin E/g DW. Cubes from wild type and GP tubers were boiled and subjected to simulated digestion to estimate bioaccessibility of carotenoids and αTC. Retention in boiled GPs exceeded 80% for β-carotene (βC), α-carotene (αC), lutein, phytoene ± and αTC, but less than 50% for phytofluene. The efficiency of partitioning of total βC, αC, E-lutein, phytoene, phytofluene and αTC in the mixed micelle fraction during small intestinal digestion was influenced by genotype, tuber content and hydrophobicity. Apical uptake of the compounds that partitioned in mixed micelles by monolayers of human intestinal Caco-2 cells during incubation for 4h was 14–20% for provitamin A and xanthophylls, 43–45% for phytoene, 23–27% for phytofluene, and 53% for αTC. These results suggest that a 150 g serving of boiled golden potatoes has the potential to contribute 42% and 23% of the daily requirement of retinol activity equivalents (RAE), as well as 34 and 17% of the daily vitamin E requirement for children and women of reproductive age, respectively.
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Skeletal muscle shows high plasticity in response to external demand. Moreover, adult skeletal muscle is capable of complete regeneration after injury, due to the properties of Muscle Stem Cells (MuSCs), the satellite cells, which follow a tightly regulated myogenic program to generate both new myofibers and new MuSCs for further needs. Whereas reactive oxygen species (ROS) and reactive nitrogen species (RNS) have long been associated with skeletal muscle physiology, their implication in the cell and molecular processes at work during muscle regeneration is more recent. This review focuses on redox regulation during skeletal muscle regeneration. An overview of the basics of ROS/RNS and antioxidant chemistry and biology occurring in skeletal muscle is first provided. Then, the comprehensive knowledge on redox regulation of MuSCs and their surrounding cell partners (macrophages, endothelial cells) during skeletal muscle regeneration is presented in normal muscle and in specific physiological (exercise-induced muscle damage, aging) and pathological (muscular dystrophies) contexts. Recent advances in the comprehension of these processes has led to the development of therapeutic assays using antioxidant supplementation, which result in inconsistent efficiency, underlying the need for new tools aimed at precisely deciphering and targeting ROS networks. This review should provide an overall insight of the redox regulation of skeletal muscle regeneration while highlighting the limits of the use of non-specific antioxidants to improve muscle function.
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Currently, cylindrical beams with radial or azimuthal polarization are being used successfully for the optical manipulation of micro- and nano-particles as well as in microscopy, lithography, nonlinear optics, materials processing, and telecommunication applications. The creation of these laser beams is carried out using segmented polarizing plates, subwavelength gratings, interference, or light modulators. Here, we demonstrate the conversion of cylindrically polarized laser beams from a radial to an azimuthal polarization, or vice versa, by introducing a higher-order vortex phase singularity. To simultaneously generate several vortex phase singularities of different orders, we utilized a multi-order diffractive optical element. Both the theoretical and the experimental results regarding the radiation transmitted through the diffractive optical element show that increasing the order of the phase singularity leads to more efficient conversation of the polarization from radial to azimuthal. This demonstrates a close connection between the polarization and phase states of electromagnetic beams, which has important implications in many optical experiments.
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Advanced glycosylation end products (AGEs) are endogenous inflammatory mediators that induce apoptosis of mesenchymal stem cells. A potential mechanism includes increased generation of reactive oxygen species (ROS). MicroRNA-223 (miR-223) is implicated in the regulation of cell growth and apoptosis in several cell types. Here, we tested the hypothesis that antioxidants N-acetylcysteine (NAC) and ascorbic acid 2-phosphate (AAP) inhibit AGE-induced apoptosis via a microRNA-dependent mechanism in human adipose tissue-derived stem cells (ADSCs). Results showed that AGE-HSA enhanced apoptosis and caspase-3 activity in ADSCs. AGE-HSA also increased ROS generation and upregulated the expression of miR-223. Interestingly, reductions in ROS generation and apoptosis, and upregulation of miR-223 were found in ADSCs treated with antioxidants NAC and AAP. Furthermore, miR-223 mimics blocked antioxidant inhibition of AGE-induced apoptosis and ROS generation. Knockdown of miR-223 amplified the protective effects of antioxidants on apoptosis induced by AGE-HSA. miR-223 acted by targeting fibroblast growth factor receptor 2. These results indicate that NAC and AAP suppress AGE-HSA-induced apoptosis of ADSCs, possibly through downregulation of miR-223.
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Purpose: The purpose of this study was to determine the therapeutic effect and mechanism of AAV-MnSOD by intravitreal injection on diabetic retinopathy (DRP) and the metabolic memory phenomenon. Methods: The effect of hyperglycemia and metabolic memory on the thickness of basement membrane, ratio of pericyte area and cross-sectional area of capillary vessels in the nerve fiber layer and outer plexiform layer; retinal capillary cell apoptosis; number of acellular capillaries and activities of retinal MnSOD and catalase were examined and compared with intravitreal injection of AAV-MnSOD by transmission electron microscopy, TUNEL assay, ELISA, and immunohistochemistry. Results: Hyperglycemia increased the thickness of capillary basement membranes in the nerve fiber layer and outer plexiform layer, decreased the ratio of pericyte area and cross-sectional area of capillary vessels, increased numbers of acellular capillaries and apoptosis of retinal capillary cells, and decreased activities of retinal MnSOD and catalase. Termination of hyperglycemia cannot reverse pathological changes listed above. Intra-vitreal injection of AAV-MnSOD dramatically elevated the level and activities of retinal MnSOD and catalase, and effectively prevented the progression of DRP and the metabolic memory phenomenon. Conclusions: Increasing reactive oxygen species concentration and continuous decreasing of antioxidant enzyme activity play important roles in DRP and the metabolic memory phenomenon. AAV-MnSOD gene therapy provides a promising strategy to inhibit this blinding disease.
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Carotenoids have been known to be effective in preventing a variety of diseases such as cancers and cardiovascular disorders. Mammalian cells cannot synthesize carotenoids and rely on a dietary source for their intake. Here we report the establishment of mammalian cells synthesizing phytoene, a kind of carotenoid, by introduction of the crtB gene, phytoene synthase gene, which was previously cloned from a bacterium, Erwinia uredovora. The phytoene-producing cells acquired a resistance against oxidative stress induced by Fe3+/adenosine 5'-diphosphate. The data suggest the feasibility of a genetic chemoprevention for oxidative stress-related disorders by endogenous synthesis of phytoene in mammalian cells.
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The Sleeping Beauty (SB) transposon is a promising technology platform for gene transfer in vertebrates; however, its efficiency of gene insertion can be a bottleneck in primary cell types. A large-scale genetic screen in mammalian cells yielded a hyperactive transposase (SB100X) with approximately 100-fold enhancement in efficiency when compared to the first-generation transposase. SB100X supported 35-50% stable gene transfer in human CD34(+) cells enriched in hematopoietic stem or progenitor cells. Transplantation of gene-marked CD34(+) cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution. In addition, SB100X supported sustained (>1 year) expression of physiological levels of factor IX upon transposition in the mouse liver in vivo. Finally, SB100X reproducibly resulted in 45% stable transgenesis frequencies by pronuclear microinjection into mouse zygotes. The newly developed transposase yields unprecedented stable gene transfer efficiencies following nonviral gene delivery that compare favorably to stable transduction efficiencies with integrating viral vectors and is expected to facilitate widespread applications in functional genomics and gene therapy.
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This study assesses consumers' willingness to purchase genetically modified (GM) food products with two different types of benefits: an input (i.e., reduced pesticides) and an output trait benefit (i.e., nutritionally enhanced). Data were collected using a telephone survey of an Italian households sample. Discrete choice approach is used to elicit the purchase intentions of the respondents. Four separate probit models are estimated to examine the effect of various factors on choices. Results suggest that majority of Italians are not willing to buy GM food products even if they are nutritionally enhanced. However, more consumers are willing to buy a nutritionally enhanced plant based GM product than a traditional plant based GM (with input trait benefit). Willingness to buy for a nutritionally enhanced animal based GM product and for a traditional animal based GM product with input trait benefit are similar. Consumers unwilling to buy GM food would not buy it even if it is nutritionally enhanced and has lower price. However, there is a niche of consumers who are willing to buy nutritionally enhanced GM food products even at a premium. Knowledge of science and trust in scientists consistently affect Italian consumers' willingness to buy the GM products.
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Rice (Oryza sativa), a major staple food, is usually milled to remove the oil-rich aleurone layer that turns rancid upon storage, especially in tropical areas. The remaining edible part of rice grains, the endosperm, lacks several essential nutrients, such as provitamin A. Thus, predominant rice consumption promotes vitamin A deficiency, a serious public health problem in at least 26 countries, including highly populated areas of Asia, Africa, and Latin America. Recombinant DNA technology was used to improve its nutritional value in this respect. A combination of transgenes enabled biosynthesis of provitamin A in the endosperm.
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The Sleeping Beauty (SB) transposon system, derived from teleost fish sequences, is extremely effective at delivering DNA to vertebrate genomes, including those of humans. We have examined several parameters of the SB system to improve it as a potential, nonviral vector for gene therapy. Our investigation centered on three features: the carrying capacity of the transposon for efficient integration into chromosomes of HeLa cells, the effects of overexpression of the SB transposase gene on transposition rates, and improvements in the activity of SB transposase to increase insertion rates of transgenes into cellular chromosomes. We found that SB transposons of about 6 kb retained 50% of the maximal efficiency of transposition, which is sufficient to deliver 70-80% of identified human cDNAs with appropriate transcriptional regulatory sequences. Overexpression inhibition studies revealed that there are optimal ratios of SB transposase to transposon for maximal rates of transposition, suggesting that conditions of delivery of the two-part transposon system are important for the best gene-transfer efficiencies. We further refined the SB transposase to incorporate several amino acid substitutions, the result of which led to an improved transposase called SB11. With SB11 we are able to achieve transposition rates that are about 100-fold above those achieved with plasmids that insert into chromosomes by random recombination. With the recently described improvements to the transposon itself, the SB system appears to be a potential gene-transfer tool for human gene therapy.
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Attempts to generate reliable and versatile vectors for gene therapy and biomedical research that express multiple genes have met with limited success. Here we used Picornavirus 'self-cleaving' 2A peptides, or 2A-like sequences from other viruses, to generate multicistronic retroviral vectors with efficient translation of four cistrons. Using the T-cell receptor:CD3 complex as a test system, we show that a single 2A peptide-linked retroviral vector can be used to generate all four CD3 proteins (CD3epsilon, gamma, delta, zeta), and restore T-cell development and function in CD3-deficient mice. We also show complete 2A peptide-mediated 'cleavage' and stoichiometric production of two fluorescent proteins using a fluorescence resonance energy transfer-based system in multiple cell types including blood, thymus, spleen, bone marrow and early stem cell progenitors.
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The symmetrically cleaving beta-carotene 15,15'-monooxygenase (BCO1) catalyzes the first step in the conversion of provitamin A carotenoids to vitamin A in the mucosa of the small intestine. This enzyme is also expressed in epithelia in a variety of extraintestinal tissues. The newly discovered beta-carotene 9',10'-monooxygenase (BCO2) catalyzes asymmetric cleavage of carotenoids. To gain some insight into the physiological role of BCO2, we determined the expression pattern of BCO2 mRNA and protein in human tissues. By immunohistochemical analysis it was revealed that BCO2 was detected in cell types that are known to express BCO1, such as epithelial cells in the mucosa of small intestine and stomach, parenchymal cells in liver, Leydig and Sertoli cells in testis, kidney tubules, adrenal gland, exocrine pancreas, and retinal pigment epithelium and ciliary body pigment epithelia in the eye. BCO2 was uniquely detected in cardiac and skeletal muscle cells, prostate and endometrial connective tissue, and endocrine pancreas. The finding that the BCO2 enzyme was expressed in some tissues and cell types that are not sensitive to vitamin A deficiency and where no BCO1 has been detected suggests that BCO2 may also be involved in biological processes other than vitamin A synthesis.
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This study investigated the effect of nutritional enhancements on consumer appeal of cultured meat. Participants (n = 775) read a passage about either nutritionally enhanced or nutritionally equivalent cultured meat, and subsequently answered questions about their beliefs, attitudes, and behavioural intentions towards cultured meat. Those in the nutritionally enhanced condition rated cultured meat as significantly healthier, more able to prevent diseases associated with meat consumption, and more likely to have benefits for society. However, they also rated cultured meat as significantly less similar to conventional meat in terms of sensory attributes including taste. This latter difference may have been caused by additional messages about taste similarity in the nutritionally equivalent condition. Overall, there was no significant difference in ratings of naturalness, overall attitudes, or purchase intent. We also found further evidence to support the existence of a large base of consumers interested in cultured meat in the US, and the higher appeal of cultured meat to liberals, meat-eaters, and those who are more familiar with the concept. The results suggest that cultured meat producers which opt to make nutritional enhancements to their products should take extra care to address consumer concerns about the possible impact on product taste, since research shows taste to be a stronger predictor of food choice than healthiness. Consumer concerns about unnaturalness, on the other hand, appear to be unaffected by nutritional enhancements.
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Cultured meat is an emerging food technology that has the potential to resolve many of the social, environmental and ethical issues surrounding traditional factory farming practices. Recently, research has begun to explore consumer attitudes to the product, revealing a number of barriers and demographic predictors. However, our understanding of the psychological mechanisms that underpin attitudes to cultured meat remains limited. In the current study, we draw on an attitude roots model (Hornsey & Fielding, 2017) to explore a range of psychological mechanisms that may underpin attitudes to cultured meat. In terms of negative attitudes and intentions toward cultured meat, the most powerful predictors were food neophobia, political conservatism, and distrust of food scientists. When it comes to absolute opposition to cultured meat - defined by the unconditional belief that it should never be allowed under any circumstances - the strongest predictors were food and hygiene disgust sensitivity subscales, food neophobia, and conspiratorial ideation. A number of presumed mechanisms held no relationships to cultured meat attitudes, including social dominance orientation, speciesism, and naturalness bias. The null results on naturalness bias are of particular interest given recent research identifying concerns about naturalness as a key barrier to consumer acceptance. These results demonstrate the need for a more nuanced understanding of the psychological mechanisms that contribute to cultured meat attitudes and engagement.
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Synthetic biology is maturing into a true engineering discipline for model microorganisms, but remains far from straightforward for most eukaryotes. Here, we outline the key challenges facing those trying to engineer biology across eukaryota and suggest areas of focus that will aid future progress.
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Cultured meat grown in-vitro from animal cells is being developed as a way of addressing many of the ethical and environmental concerns associated with conventional meat production. As commercialisation of this technology appears increasingly feasible, there is growing interest in the research on consumer acceptance of cultured meat. We present a systematic review of the peer-reviewed literature, and synthesize and analyse the findings of 14 empirical studies. We highlight demographic variations in consumer acceptance, factors influencing acceptance, common consumer objections, perceived benefits, and areas of uncertainty. We conclude by evaluating the most important objections and benefits to consumers, as well as highlighting areas for future research.
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Rational approaches to modifying cells to make molecules of interest are of substantial economic and scientific interest. Most of these efforts aim at the production of native metabolites, expression of heterologous biosynthetic pathways, or protein expression. Reviews of these topics have largely focused on individual strategies or cell types, but collectively they fall under the broad umbrella of a growing field known as cell factory engineering. Here we condense >130 reviews and key studies in the art into a meta-review of cell factory engineering. We identified 33 generic strategies in the field, all applicable to multiple types of cells and products, and proven successful in multiple major cell types. These apply to three major categories: production of native metabolites and/or bioactives, heterologous expression of biosynthetic pathways, and protein expression. This meta-review provides general strategy guides for the broad range of applications of rational engineering of cell factories.
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Background Carotenoids are C-30 or C-40 based pigments with antioxidant/anti-inflammatory properties, some possessing vitamin A activity. Their dietary intake, especially within fruits and vegetables, has been associated with a decreased risk of chronic diseases, including type-2 diabetes, cardiovascular diseases, age-related macular degeneration, and several types of cancer. However, their bioavailability is wide-ranging and is affected by numerous factors. Recent findings showing that the intestinal absorption of carotenoids involves proteins have raised new relevant questions about factors that can affect their bioavailability. It is therefore opportune to present a current overview of this topic. Scope and approach This review begins by exploring what is known, as well as what is unknown, about the metabolism of carotenoids in the human upper gastrointestinal tract and then presents a methodical evaluation of factors assumed to affect carotenoid bioavailability. Key findings and conclusions Numerous unanswered questions remain about the metabolism of carotenoids in the intestinal lumen and about the factors affecting their absorption efficiency. These gaps need to be filled to be able to better understand the variability of individual responses to these compounds so as to promote guidelines towards personalized dietary recommendations in order to increase carotenoid absorption efficiency and hence their health effects. Two main conclusions can be drawn. First, the efficiency of carotenoid absorption is affected by several dietary factors (e.g. food matrix, fat, and fat-soluble micronutrients). Second, carotenoid bioavailability also depends on host-related factors, e.g. diseases, life-style habits, gender and age, as well as genetic variations such as single nucleotide polymorphisms.
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Red meat (beef, veal, pork, lamb and mutton) consumption contributes several important nutrients to the diet, for example essential amino acids, vitamins (including B12) and minerals (including iron and zinc). Processed red meat (ham, sausages, bacon, frankfurters, salami, etc.) undergoes treatment (curing, smoking, salting or the use of chemical preservatives and additives) to improve its shelf life and/or taste. During recent decades, consumption of red meat has been increasing globally, especially in developing countries. At the same time, there has been growing evidence that high consumption of red meat, especially of processed meat, may be associated with an increased risk of several major chronic diseases. Here, a comprehensive summary is provided of the accumulated evidence based on prospective cohort studies regarding the potential adverse health effects of red meat consumption on major chronic diseases, such as diabetes, coronary heart disease, heart failure, stroke and cancer at several sites, and mortality. Risk estimates from pooled analyses and meta-analyses are presented together with recently published findings. Based on at least six cohorts, summary results for the consumption of unprocessed red meat of 100 g day−1 varied from nonsignificant to statistically significantly increased risk (11% for stroke and for breast cancer, 15% for cardiovascular mortality, 17% for colorectal and 19% for advanced prostate cancer); for the consumption of 50 g day−1 processed meat, the risks were statistically significantly increased for most of the studied diseases (4% for total prostate cancer, 8% for cancer mortality, 9% for breast, 18% for colorectal and 19% for pancreatic cancer, 13% for stroke, 22% for total and 24% for cardiovascular mortality and 32% for diabetes). Potential biological mechanisms underlying the observed risks and the environmental impact of red meat production are also discussed. The evidence-based integrated message is that it is plausible to conclude that high consumption of red meat, and especially processed meat, is associated with an increased risk of several major chronic diseases and preterm mortality. Production of red meat involves an environmental burden. Therefore, some European countries have already integrated these two issues, human health and the ‘health of the planet’, into new dietary guidelines and recommended limiting consumption of red meat.
Article
Clustered regularly-interspaced short palindromic repeat (CRISPR) sequences cooperate with CRISPR-associated (Cas) proteins to form the basis of CRISPR-Cas adaptive immune systems in prokaryotes. For more than 20 years, these systems were of interest only to specialists, mainly molecular microbiologists, who tried to understand the properties of this unique defense mechanism. In 2012, the potential of CRISPR-Cas systems was uncovered and these were presented as genome-editing tools with an outstanding capacity to trigger targeted genetic modifications that can be applied to virtually any organism. Shortly thereafter, in early 2013, these tools were shown to efficiently drive specific modification of mammalian genomes. This review attempts to summarize, in a comprehensive manner, the key events and milestones that brought CRISPR-Cas technology from prokaryotes to mammals.
Article
Synthetic biology is a promising multidisciplinary field that brings together experts in scientific disciplines from cell biology to engineering with the goal of constructing elements that do not occur in nature for use in various applications, such as the development of novel approaches to improving healthcare management. Current disease treatment strategies are typically based on the diagnosis of phenotypic changes, which are often the result of an accumulation of endogenous metabolic defects in the human body. These defects occur when the tight regulation of physiological processes is disturbed by genetic alterations, protein function losses, or environmental changes. Such disturbances can result in the development of serious disorders that are often associated with aberrant physiological levels of certain biomolecules (e.g., metabolites, cytokines, and growth factors), which may lead to specific pathogenic states. However, these aberrant levels can also serve as biomarkers for the precise detection and specification of disease types. Clinical interventions are often conducted during the late stages of disease pathogenesis because of a lack of early detection of these physiological disturbances, which results in disease treatment rather than prevention. Therefore, advanced therapeutic tools must be developed to link therapeutic intervention to early diagnosis. Recent advances in the field of synthetic biology have enabled the design of complex gene circuits that can be linked to a host's metabolism to autonomously detect disease‐specific biomarkers and then reprogrammed to produce and release therapeutic substances in a self‐sufficient and automatic fashion, thereby restoring the physiological balance of the host and preventing the progression of the disease. This concept offers a unique opportunity to design treatment protocols that could replace conventional strategies, especially for diseases with complex and recurrent dynamics, such as chronic diseases. WIREs Syst Biol Med 2016, 8:402–422. doi: 10.1002/wsbm.1345 This article is categorized under: • Translational, Genomic, and Systems Medicine > Diagnostic Methods • Translational, Genomic, and Systems Medicine > Therapeutic Methods • Translational, Genomic, and Systems Medicine > Translational Medicine
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Cultured, or in vitro, meat consists of edible biomass grown from animal stem cells in a factory, or carnery. In the coming decades, in vitro biomass cultivation could enable the production of meat without the need to raise livestock. Using an anticipatory life cycle analysis framework, the study described herein examines the environmental implications of this emerging technology and compares the results with published impacts of beef, pork, poultry, and another speculative analysis of cultured biomass. While uncertainty ranges are large, the findings suggest that in vitro biomass cultivation could require smaller quantities of agricultural inputs and land than livestock; however, those benefits could come at the expense of more intensive energy use as biological functions such as digestion and nutrient circulation are replaced by industrial equivalents. From this perspective, large-scale cultivation of in vitro meat and other bioengineered products could represent a new phase of industrialization with inherently complex and challenging trade-offs.
Article
For thousands of years, yeast has been used for making beer, bread, and wine. In modern times, it has become a commercial workhorse for producing fuels, chemicals, and pharmaceuticals such as insulin, human serum albumin, and vaccines against hepatitis virus and human papillomavirus. Yeast has also been engineered to make chemicals at industrial scale (e.g., succinic acid, lactic acid, resveratrol) and advanced biofuels (e.g., isobutanol) ( 1 ). On page 1095 of this issue, Galanie et al. ( 2 ) demonstrate that yeast can now be engineered to produce opioids ( 2 ), a major class of compounds used for treating severe pain. Their study represents a tour de force in the metabolic engineering of yeast, as it involved the expression of genes for more than 20 enzymatic activities from plants, mammals, bacteria, and yeast itself. It clearly represents a breakthrough advance for making complex natural products in a controlled and sustainable way.
Article
Corynebacterium glutamicum is the workhorse of biotechnological amino acid production. For more than 50 years amino acid producing strains of this actinomycete have been improved by classical breeding, metabolic engineering and systems and synthetic biology approaches. This review focusses mainly on recent developments on C. glutamicum strain development for non-natural products. Recently, metabolite sensors have accelerated classical strain breeding. Synthetic pathways for access to alternative carbon sources, such as pentoses, and to new products, such as α, ω-amino acids, α, ω-diamines, α-keto acids, isobutanol, carotenoids and terpenes, have been embedded in the central metabolism of C. glutamicum. Furthermore, C. glutamicum is a chassis for new and improved production processes that has been improved in two ways: by rendering it biotin prototrophic and by curing it from its prophage DNA followed by further genome reduction. The first combinations of this chassis approach with production will be highlighted. Although their transfer to industrial scale processes will have to be evaluated, these recent achievements indicate how synthetic biology helps realizing proof-of-principles. Moreover, current and future synthetic biology technology developments hold the promise to explore the full potential of C. glutamicum as production host for value-added chemicals. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Article
The EU Sixth Framework Programme Integrated Project 'Pharma-Planta' developed an approved manufacturing process for recombinant plant-made pharmaceutical proteins (PMPs) using the human HIV-neutralizing monoclonal antibody 2G12 as a case study. In contrast to the well-established Chinese hamster ovary platform, which has been used for the production of therapeutic antibodies for nearly 30 years, only draft regulations were initially available covering the production of recombinant proteins in transgenic tobacco plants. Whereas recombinant proteins produced in animal cells are secreted into the culture medium during fermentation in bioreactors, intact plants grown under nonsterile conditions in a glasshouse environment provide various 'plant-specific' regulatory and technical challenges for the development of a process suitable for the acquisition of a manufacturing licence for clinical phase I trials. During upstream process development, several generic steps were addressed (e.g. plant transformation and screening, seed bank generation, genetic stability, host plant uniformity) as well as product-specific aspects (e.g. product quantity). This report summarizes the efforts undertaken to analyse and define the procedures for the GMP/GACP-compliant upstream production of 2G12 in transgenic tobacco plants from gene to harvest, including the design of expression constructs, plant transformation, the generation of production lines, master and working seed banks and the detailed investigation of cultivation and harvesting parameters and their impact on biomass, product yield and intra/interbatch variability. The resulting procedures were successfully translated into a prototypic manufacturing process that has been approved by the German competent authority. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.
Article
Stable gene expression in mammalian cells is a prerequisite for many in vitro and in vivo experiments. However, either the integration of plasmids into mammalian genomes or the use of retro-/lentiviral systems have intrinsic limitations. The use of transposable elements, e.g. the Sleeping Beauty system (SB), circumvents most of these drawbacks (integration sites, size limitations) and allows the quick generation of stable cell lines. The integration process of SB is catalyzed by a transposase and the handling of this gene transfer system is easy, fast and safe. Here, we report our improvements made to the existing SB vector system and present two new vector types for robust constitutive or inducible expression of any gene of interest. Both types are available in 16 variants with different selection marker (puromycin, hygromycin, blasticidin, neomycin) and fluorescent protein expression (GFP, RFP, BFP) to fit most experimental requirements. With this system it is possible to generate cell lines from stable transfected cells quickly and reliably in a medium-throughput setting (3-5 days). Cell lines robustly express any gene-of-interest, either constitutively or tightly regulated by doxycycline. This allows many laboratory experiments to speed up generate data in a rapid and robust manner. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Article
Free radical scavenging properties of Lycopene (LYC), Phytofluene (PF) and Phytoene (PE) are studied experimentally, considering the electron transfer mechanism and using the ABTS•+ decolouration assay. Density Functional Theory is used to obtain the theoretical UV-visible spectra of ABTS (neutral and cation) and to determine the free radical scavenger capacity in terms of the electron donor mechanism and the deactivation of singlet oxygen. The experimental data agreed very well with the theoretical results. Considering both mechanisms, LYC, PF and PE can be considered good free radical scavengers, with LYC turning out to be the best and PE the worst antiradical. However, the triplets that formed following the deactivation of singlet oxygen presented similar capacity for donating electrons. In the case of triplets, the antiradical capacity of LYC, PF and PE is similar in terms of electron donor mechanism. Although the results indicated that PF and PE are not as effective antiradicals as LYC, to be expected due to the number of conjugated double bonds, they present higher antioxidant capacity considering the small number of conjugated double bonds.
Article
Geometrical isomers of carotenoids behave differently in aspects like stability towards oxidants, bioavailability, vitamin A activity and specificity for enzymes. The availability of HPLC methods for their detailed profiling is therefore advisable to expand our knowledge on their metabolism and biological role. In this paper the development of a methodology to determine the highest number of geometrical isomers of major carotenoids in humans (phytoene, phytofluene, lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene and lycopene) is described. To assess its usefulness with biological samples both postprandial human plasma and lung samples from ferrets were analysed. Up to 48 isomers of the main human carotenoids were separated in 62min. This is to the best of our knowledge the report of the highest number of carotenoid geometrical isomers separated with a HPLC method. Twenty-six different carotenoid isomers were readily detected in the biological samples.
Article
Ribeye steaks (Longissimus muscle) and ground beef from 15 Angus or Angus × Hereford steers slaughtered at weights of 480–500 kg were evaluated for proximate composition, color, β-carotene content and consumer preference. Three groups of five animals were finished on annual ryegrass pasture (Lolium multiflorum), ryegrass and ‘Coastal’ bermu-dagrass hay (Cynodon hybrid), or a feedlot diet.β-carotene content of ribeye steaks and ground beef was higher (p < 0.05) for the forage finished animals than those finished in the feedlot. There was no difference in scores from consumer panels (n = 80) for steaks from feedlot or pasture finished animals, but scores for ground beef from cattle finished on the feedlot diet were higher than other treatments (p < 0.05).
Article
Carotenoids participate in light harvesting and are essential for photoprotection in photosynthetic plant tissues. They also furnish non-photosynthetic flowers and fruits with yellow to red colors to attract animals for pollination and dispersal of seeds. Although animals can not synthesize carotenoids de novo, carotenoid-derived products such as retinoids (including vitamin A) are required as visual pigments and signaling molecules. Dietary carotenoids also provide health benefits based on their antioxidant properties. The main pathway for carotenoid biosynthesis in plants and microorganisms has been virtually elucidated in recent years, and some of the identified biosynthetic genes have been successfully used in metabolic engineering approaches to overproduce carotenoids of interest in plants. Alternative approaches that enhance the metabolic flux to carotenoids by upregulating the production of their isoprenoid precursors or interfere with light-mediated regulation of carotenogenesis have been recently shown to result in increased carotenoid levels. Despite spectacular achievements in the metabolic engineering of plant carotenogenesis, much work is still ahead to better understand the regulation of carotenoid biosynthesis and accumulation in plant cells. New genetic and genomic approaches are now in progress to identify regulatory factors that might significantly contribute to improve the nutritional value of plant-derived foods by increasing their carotenoid levels.
Article
As one of the alternatives for livestock meat production, in vitro culturing of meat is currently studied. The generation of bio-artificial muscles from satellite cells has been ongoing for about 15 years, but has never been used for generation of meat, while it already is a great source of animal protein. In order to serve as a credible alternative to livestock meat, lab or factory grown meat should be efficiently produced and should mimic meat in all of its physical sensations, such as visual appearance, smell, texture and of course, taste. This is a formidable challenge even though all the technologies to create skeletal muscle and fat tissue have been developed and tested. The efficient culture of meat will primarily depend on culture conditions such as the source of medium and its composition. Protein synthesis by cultured skeletal muscle cells should further be maximized by finding the optimal combination of biochemical and physical conditions for the cells. Many of these variables are known, but their interactions are numerous and need to be mapped. This involves a systematic, if not systems, approach. Given the urgency of the problems that the meat industry is facing, this endeavor is worth undertaking. As an additional benefit, culturing meat may provide opportunities for production of novel and healthier products.
Article
Limiting ergosterol accumulation through metabolic control increased lycopene production by Blakeslea trispora. Lycopene and ergosterol are both biosynthesized from a common precursor, farnesyl diphosphate (FPP). The effects of two ergosterol biosynthesis inhibitors, terbinafine hydrochloride (TH) and ketoconazole, on the production of lycopene by B. trispora were investigated. TH at 0.7 mg/l and ketoconazole at 30 mg/l added to the medium at 48 h of fermentation caused an increase in lycopene content of 23% or 277%, respectively. The timing of addition for both inhibitors at 48 h resulted in the most optimal lycopene productivity, however, compared with TH, ketoconazole was superior in enhancing lycopene production by inhibiting ergosterol biosynthesis.
Article
Mitochondrial reactive oxygen species (mROS) have been considered detrimental to cells. However, their physiological roles as signaling mediators have not been thoroughly explored. Here, we investigated whether mROS generated from mitochondrial electron transport chain (mETC) complex I stimulated muscle differentiation. Our results showed that the quantity of mROS was increased and that manganese superoxide dismutase (MnSOD) was induced via NF-κB activation during muscle differentiation. Mitochondria-targeted antioxidants (MitoQ and MitoTEMPOL) and mitochondria-targeted catalase decreased mROS quantity and suppressed muscle differentiation without affecting the amount of ATP. Mitochondrial alterations, including the induction of mitochondrial transcription factor A and an increase in the number and size of mitochondria, and functional activations were observed during muscle differentiation. In particular, increased expression levels of mETC complex I subunits and a higher activity of complex I than other complexes were observed. Rotenone, an inhibitor of mETC complex I, decreased the mitochondrial NADH/NAD(+) ratio and mROS levels during muscle differentiation. The inhibition of complex I using small interfering RNAs and rotenone reduced mROS levels, suppressed muscle differentiation, and depleted ATP levels with a concomitant increase in glycolysis. From these results, we conclude that complex I-derived O(2)·(-), produced through reverse electron transport due to enhanced metabolism and a high activity of complex I, was dismutated into H(2)O(2) by MnSOD induced via NF-κB activation and that the dismutated mH(2)O(2) stimulated muscle differentiation as a signaling messenger.
Article
Metabolic engineering has the potential to produce from simple, readily available, inexpensive starting materials a large number of chemicals that are currently derived from nonrenewable resources or limited natural resources. Microbial production of natural products has been achieved by transferring product-specific enzymes or entire metabolic pathways from rare or genetically intractable organisms to those that can be readily engineered, and production of unnatural specialty chemicals, bulk chemicals, and fuels has been enabled by combining enzymes or pathways from different hosts into a single microorganism and by engineering enzymes to have new function. Whereas existing production routes use well-known, safe, industrial microorganisms, future production schemes may include designer cells that are tailor-made for the desired chemical and production process. In any future, metabolic engineering will soon rival and potentially eclipse synthetic organic chemistry.
Article
Cell-based biosensors (CBBs) have emerged as powerful functional tools for the rapid detection of hazards and threats associated with food, agriculture, environment and biosecurity. CBBs detect the functional aspects of a host-hazard interaction and render an accurate estimation of the risks. Assessing hazard-induced physiological responses, such as receptor-ligand interactions, signal transduction, gene expression, membrane damage, apoptosis and oncosis of living sensing organisms can provide insight into the basis of toxicity for a particular hazard. This review highlights the progress made in developing mammalian CBBs for pathogens and toxins, with special emphasis on multidisciplinary approaches that combine molecular biology and microbiology with methods used in physics and engineering, which led to the development of a three-dimensional cell-culture system and high-throughput screening employing optical and electrical systems.
Article
THE muscular dystrophies are a group of hereditary disorders manifested by a progressive wasting of the skeletal muscles. In spite of extensive studies, the nature of the primary lesion is unknown (for review see ref. 1). Because of the complex interaction between tissues, it is difficult to study this question in vivo. Therefore attempts have been made to investigate this question in cultures of dystrophic muscles of human or animal origin. Tissue explants as well as monolayer primary cell cultures contain, in addition to the myogenic cells, a heterogeneous cell population, the composition of which might differ in normal and dystrophic muscle cultures. It is difficult in such experiments to distinguish between properties intrinsic to the myogenic cells and effects exerted by other cell types. Indeed, previous experiments have yielded conflicting conclusions2-6. We therefore tested the possibility of obtaining cell cultures consisting of pure populations of myogenic cells obtained from dystrophic muscles. The present report describes the isolation of a cloned population of such cells, derived from adult dystrophic mouse muscle, that can proliferate and differentiate in cell culture.
Article
Sleeping Beauty (SB), a member of the Tc1/mariner superfamily of transposable elements, is the only active DNA-based transposon system of vertebrate origin that is available for experimental manipulation. We have been using the SB element as a research tool to investigate some of the cis and trans-requirements of element mobilization, and mechanisms that regulate transposition in vertebrate species. In contrast to mariner transposons, which are regulated by overexpression inhibition, the frequency of SB transposition was found to be roughly proportional to the amount of transposase present in cells. Unlike Tc1 and mariner elements, SB contains two binding sites within each of its terminal inverted repeats, and we found that the presence of both of these sites is a strict requirement for mobilization. In addition to the size of the transposon itself, the length as well as sequence of the DNA outside the transposon have significant effects on transposition. As a general rule, the closer the transposon ends are, the more efficient transposition is from a donor molecule. We have found that SB can transform a wide range of vertebrate cells from fish to human. However, the efficiency and precision of transposition varied significantly among cell lines, suggesting potential involvement of host factors in SB transposition. A positive-negative selection assay was devised to enrich populations of cells harboring inserted transposons in their chromosomes. Using this assay, of the order of 10,000 independent transposon insertions can be generated in human cells in a single transfection experiment. Sleeping Beauty can be a powerful alternative to other vectors that are currently used for the production of transgenic animals and for human gene therapy.
Article
The paired box transcription factor Pax7 was isolated by representational difference analysis as a gene specifically expressed in cultured satellite cell-derived myoblasts. In situ hybridization revealed that Pax7 was also expressed in satellite cells residing in adult muscle. Cell culture and electron microscopic analysis revealed a complete absence of satellite cells in Pax7(-/-) skeletal muscle. Surprisingly, fluorescence-activated cell sorting analysis indicated that the proportion of muscle-derived stem cells was unaffected. Importantly, stem cells from Pax7(-/-) muscle displayed almost a 10-fold increase in their ability to form hematopoietic colonies. These results demonstrate that satellite cells and muscle-derived stem cells represent distinct cell populations. Together these studies suggest that induction of Pax7 in muscle-derived stem cells induces satellite cell specification by restricting alternate developmental programs.
Article
Transgenic plants are showing considerable potential for the economic production of proteins, with a few already being marketed. Recent clinical trials of pharmaceuticals produced from transgenic plants are encouraging, with plant glycans showing reassuringly poor immunogenicity. Our increasing understanding of protein targeting and accumulation should further improve the potential of this new technology.
Article
The large number of candidate genes made available by comprehensive genome analysis requires that relatively rapid techniques for the study of function be developed. Here, we report a rapid and convenient electroporation method for both gain- and loss-of-function studies in vivo and in vitro in the rodent retina. Plasmid DNA directly injected into the subretinal space of neonatal rodent pups was taken up by a significant fraction of exposed cells after several pulses of high voltage. With this technique, GFP expression vectors were efficiently transfected into retinal cells with little damage to the operated pups. Transfected GFP allowed clear visualization of cell morphologies, and the expression persisted for at least 50 days. DNA-based RNA interference vectors directed against two transcription factors important in photoreceptor development led to photoreceptor phenotypes similar to those of the corresponding knockout mice. Reporter constructs carrying retinal cell type-specific promoters were readily introduced into the retina in vivo, where they exhibited the appropriate expression patterns. Plasmid DNA was also efficiently transfected into retinal explants in vitro by high-voltage pulses.
Article
The NF-kappaB/iNOS pathway stimulates muscle differentiation downstream of the PI 3-kinase/p38 MAPK pathway and diverse antioxidants block muscle differentiation. Therefore, we here investigated whether Nox 2 links those two myogenic pathways in H9c2 and C2C12 myoblasts. Compared with the proliferation stage, ROS generation was enhanced from the early stage of differentiation and gradually increased as differentiation progressed. Antioxidants suppressed the activated NF-kappaB/iNOS pathway during muscle differentiation. Nox 2 activity was also increased during muscle differentiation. Treatment with DPI and apocynin, two inhibitors of NADPH oxidase, and suppression of Nox 2 expression using siRNA, but not Nox 1, inhibited NADPH oxidase activity, muscle differentiation, and the NF-kappaB/iNOS pathway. Inhibition of PI 3-kinase and p38 MAPK suppressed the Nox 2/NF-kappaB/iNOS pathway. Nitric oxide restored muscle differentiation blocked by treatment with antioxidants or suppression of the Nox 2/NF-kappaB/iNOS pathway. In conclusion, Nox 2 stimulates muscle differentiation downstream of the PI 3-kinase/p38 MAPK pathway by activating the NF-kappaB/iNOS pathway via ROS generation.
Article
Flavonoids are potent scavengers of reactive oxygen species (ROS) that effectively prevent erythrocyte oxidation. Their antioxidant activities are governed by their structural characteristics and their ability to interact with and penetrate lipid bilayers. In order to gain a better understanding of the relationship between cholesterol contents and the antioxidant effectiveness of flavonoids against oxidative damage induced by ROS in cells, here we analyzed the integrity and structural stability of cholesterol-modified (enriched or depleted) and control erythrocytes exposed to tert-butyl hydroperoxide in the presence of quercetin or rutin. In control and cholesterol-enriched erythrocytes, quercetin provided greater protection against lipid peroxidation, ROS formation, and it preserved better cellular integrity than rutin. Both antioxidants suppressed the alterations in membrane fluidity and lipid losses with similar efficiency, reducing hemoglobin oxidation by 30% and GSH losses by 60% in the above-mentioned erythrocytes. Cholesterol depletion reduced the efficiency of the antioxidant power of both flavonoids against oxidative damage induced in the erythrocyte membrane, while a stronger degree of protection of GSH and hemoglobin contents was observed, mainly in the presence of rutin. These findings suggest a preferential incorporation of the antioxidants into the membranes from erythrocytes with normal and high cholesterol contents, whereas they would mainly be located in the cytoplasm of cholesterol-depleted erythrocytes.
Article
Electrically excitable cells are important in the normal functioning and in the pathophysiology of many biological processes. These cells are typically embedded in dense, heterogeneous tissues, rendering them difficult to target selectively with conventional electrical stimulation methods. The algal protein Channelrhodopsin-2 offers a new and promising solution by permitting minimally invasive, genetically targeted and temporally precise photostimulation. Here we explore technological issues relevant to the temporal precision, spatial targeting and physiological implementation of ChR2, in the context of other photostimulation approaches to optical control of excitable cells.
Formal agreement between FDA and USDA regarding oversight of human food produced using animal cell technology derived from cell lines of USDAamenable species. WWW Document
FDA, 2019. Formal agreement between FDA and USDA regarding oversight of human food produced using animal cell technology derived from cell lines of USDAamenable species. WWW Document]. URL. https://www.fda.gov/food/domest ic-interagency-agreements-food/formal-agreement-between-fda-and-usda-regarding -oversight-human-food-produced-using-animal-cell (accessed 4.6.20).